Calpain-1 Signaling Pathways in Platelets

说明(申请人提供)：钙蛋白酶是一种半胱氨酸蛋白酶，与多种细胞过程有关，如细胞骨架组织、细胞增殖、细胞凋亡、细胞运动和止血。我们建议在以下特定目的下验证Calain-1通过调节PTP1B的活性来调节血小板信号转导的假说：(1)Calain-1调节PTP1B。我们将使用区域特异性抗体和免疫复合体磷酸酶分析来确定PTP1B在野生型和Calain-1缺失的小鼠血小板激活时的裂解状态。我们将检测PTP1B的降解和降解片段的比活性，并研究PTP1B对血小板c-Src激活的影响。这些研究将有助于阐明Calain-1在调节PTP1B中的作用，并为Calain-I缺失的小鼠血小板中酪氨酸磷酸酶活性增加提供解释。(2)依赖Calain-1的β-3整合素与血小板细胞骨架相互作用的机制。我们计划通过凝胶电泳法和质谱法确定血小板中PTP1B底物的特性。使用携带747和759位酪氨酸保守和自由基取代的β-3整合素细胞质结构域，我们将确定它们的磷酸化化学计量、对PTP1B去磷酸化的敏感性，并量化它们与Shc、肌球蛋白和新发现的PTP1B底物的相互作用。在Biacore技术的帮助下，这些实验将有助于阐明PTP1B的下游效应器，这些效应器对血小板由外向内的信号传递至关重要。(3)Calain-1信号在血小板扩散中的作用。利用具有纯C57BL/6J遗传背景的calain-1基因缺失小鼠，我们将通过鉴定小鼠血小板中潜在的PTP1B非依赖底物来研究促进血小板扩散的机制。我们还将测试具有纯C57BL/6J遗传背景的calain-1基因缺失小鼠是否在血小板分泌和体内血栓形成状态方面显示异常。总之，这些研究将有助于阐明Calain-1在整合血小板信号的共同和不同方面的作用，揭示PTP1B调节的新方面，并提供对血小板聚集和凝块回缩途径机制的更好理解。最终，这些研究可能会导致开发新的治疗策略来对抗血栓形成。公共卫生相关性：钙蛋白酶是一种半胱氨酸蛋白酶，与多种细胞过程有关，如细胞增殖、细胞凋亡、细胞运动和止血。拟议的研究将阐明Calain-1在血小板信号转导中的作用，并提供更好的了解血小板聚集和凝块回缩途径的机制。最终，这些研究可能会导致开发新的治疗策略来对抗血栓形成。

DESCRIPTION (provided by applicant): Calpains are cysteine proteases implicated in a wide variety of cellular processes such as cytoskeletal organization, cell proliferation, apoptosis, cell motility, and hemostasis. We propose to test the hypothesis that calpain-1 regulates platelet signaling by modulating the activity of PTP1B under the following specific aims: (1) Regulation of PTP1B by calpain-1. We will determine the status of PTP1B cleavage in the wild type and calpain-1 null mouse platelets upon activation using domain-specific antibodies and immune complex phosphatase assays. We will measure the degradation of PTP1B and specific activity of degraded fragments and investigate the effect of PTP1B on c-Src activation in platelets. These studies will help to clarify the role of calpain-1 in the regulation of PTP1B and provide an explanation for the increased tyrosine phosphatase activity in calpain-I null mouse platelets. (2) Mechanism of calpain-1 dependent beta-3 integrin interactions with the platelet cytoskeleton. We plan to determine the identity of PTP1B substrates in platelets by gel electrophoresis and mass spectrometry. Using beta-3 integrin cytoplasmic domain constructs carrying conservative and radical replacement of tyrosines at 747 and 759, we will determine their phosphorylation stoichiometry, susceptibility to dephosphorylation by PTP1B, and quantify their interactions with Shc, myosin, and newly identified PTP1B substrates. These experiments, assisted by the BIAcore technology, will help to clarify the downstream effectors of PTP1B that are critical for outside-in signaling in platelets. (3) Role of calpain-1 signaling in platelet spreading. Using calpain-1 null mice with pure C57BL/6J genetic background, we will investigate the mechanism of enhanced platelet spreading by identifying the potential PTP1B-independent substrates of calpain-1 in mouse platelets. We will also test whether the calpain-1 null mice with the pure C57BL/6J genetic background display abnormalities in the platelet secretion and in vivo thrombosis status. Together, these studies will help to clarify the role of calpain-1 in integrating common and divergent aspects of platelet signaling, reveal novel aspects of PTP1B regulation, and provide a better understanding of the mechanism of platelet aggregation and clot retraction pathways. Ultimately, these studies may lead to the development of new therapeutic strategies against thrombosis. PUBLIC HEALTH RELEVANCE: Calpains are cysteine proteases implicated in a wide variety of cellular processes such as cell proliferation, apoptosis, cell motility, and hemostasis. The proposed studies will clarify the role of calpain-1 in platelet signaling and provide a better understanding of the mechanism of platelet aggregation and clot retraction pathways. Ultimately, these studies may lead to the development of new therapeutic strategies against thrombosis.